An example of a so-called "deficient selection marker needed for the growth of the yeast" is the LEU2d gene described by Kingsman c.s. (reference 1), who described the development of a multicopy integrative vector which was dispersed throughout the genome using the transposable Ty element Ty1-15 (reference 2). The element was engineered to contain two selectable markers, TRP1 (reference 3) and LEU2 from pMA3a, and the PGK expression signals from pMA91 (reference 4) with an IFN-.alpha..sub.2 coding sequence (reference 5). A single copy of the engineered Ty was integrated into the genome using a linear fragment to stimulate recombination across the ends of the element and thereby replacing an endogenous element. Transformants were selected for the TRP1 marker. Few transformants were obtained by selecting for LEU2 as insufficient enzyme was produced by a single copy of this gene. The transformant was then grown in decreasing concentrations of leucine to select for an increase in the copy number of the LEU2 gene, presumably by spread of the Ty element throughout the genome by gene conversion and transposition (reference 6). A strain was constructed which produced 8.times.10.sup.5 molecules of IFN per cell; this being intermediate between yields from single copy ARS/CEN vectors (10.sup.5 molecules/cell) and from multicopy vectors such as pMA91 (6.times.10.sup.6 molecules/cell).
For a practical stable production system with a transformed yeast the use of Ty elements has certain disadvantages.
Therefore a need exists for other systems by which multicopy integration of heterologous genes in fungi such as yeast and moulds can be achieved.